JP2019511219A - Method of differentiating pluripotent stem cells - Google Patents

Abstract

An object of the present invention is to provide a method for differentiating pluripotent stem cells. A method of differentiating pluripotent stem cells, comprising the steps of: seeding pluripotent stem cells in a container provided with a seeding medium; differentiating said pluripotent stem cells in said container; and wherein said pluripotent stem cells are precursors Upon reaching the stage, transferring the pluripotent stem cells from the container into the chamber of the bioreactor; and maturing the pluripotent stem cells in the chamber, the floor of the chamber being concave or convex The fluid of the culture medium flows in the chamber and oxygen is supplied to the chamber.
[Selected figure] Figure 1

Description

  The present invention relates to methods of differentiating pluripotent stem cells.

  Traditionally, liver cell lines, primary cells and / or animal models for demonstrating cellular and molecular mechanisms involved in the origin of liver disease, for the development of new treatments or for toxicological risk assessment of substances Was used. The results obtained using these different models can be criticized for several reasons. The most commonly used cell lines are those generated or immortalized by cancer and carry genetic mutations that lead to deregulation of the major signaling pathways. Animal models are not at all sufficient for housing cost reasons and for ethical reasons. More importantly, animals are not a good model for pharmacological toxicology studies, as about 50% toxicity of the drug can be effectively predicted. If primary hepatocytes are currently the reference model for studies of liver metabolism in vitro, it also indicates a number of limitations. The number of donors is limited and the persistence / quality of the obtained hepatocytes is low. Furthermore, hepatocyte batch variability associated with genetic polymorphism donors complicates standardized testing. In particular, the last point is that hepatocytes do not grow in culture and lose substantially irreversibly in culture. Development of alternative sources of mature and functional hepatocytes is essential.

  Hepatocytes differentiated from pluripotent stem cells have emerged as a promising source. Pluripotent stem cells have two main properties, and pluripotent stem cells may differentiate into all types of cells that make up the body (pluripotent) and in ad hoc conditions It can multiply indefinitely (self-regeneration). These cells represent a potentially inexhaustible source of mature, functionally differentiated cells (see, eg, Non-Patent Document 1).

R. E. Schwartz, et al. Pluripotent stem cell-derived hepatocyte-like cells. Biotechnology Advances 32 (2014): 504-513.

  However, the aforementioned approach is not yet fully satisfactory. HEP-LC (Hepatocytes Like Cells) still represent a primitive differentiation pattern (AFP, SOX17) indicating that maturation is not complete. Different hypotheses can be made to account for the immaturity of these cells: (i) the absence of interactions between these cells and other component hepatocytes; (ii) within the system used Lack of hemodynamic stress; (iii) Lack of organ interaction; (iv) weak concentrations of autocrine and paracrine elements in cell culture medium.

  The present invention aims to provide a method for differentiating pluripotent stem cells.

  Thus, the present disclosure is a method of differentiating pluripotent stem cells: seeding pluripotent stem cells in a container comprising a seeding medium; differentiating pluripotent stem cells in said container; Transferring the pluripotent stem cells from the container into the chamber of the bioreactor when the pluripotent stem cells reach the precursor stage; and maturing the pluripotent stem cells in the chamber, the floor of the chamber Provides a method comprising a recess or a protrusion, wherein the fluid of the culture medium flows in the chamber and oxygen is supplied to the chamber.

  In another method, the bioreactor is placed in a perfusion loop and connected to a pump in the perfusion loop, and the perfusion loop is filled with media.

  In yet another method, the bottom of the chamber includes an array of microchambers and microchannels.

  In still another method, the pluripotent stem cells are human induced pluripotent stem cells.

  In still another method, the human induced pluripotent stem cells are differentiated into HEP-LC.

  In still another method, the precursor stage is a stage in which definitive endoderm is formed, a stage in which a specific liver pattern is formed, or a stage in which immature hepatoblasts are formed.

  In yet another method, the human induced pluripotent stem cells are transferred from the container into the chamber of the bioreactor, with all sub-types of immature liver-like cells adhered together.

  In yet another method, the human induced pluripotent stem cells are matured in the chamber of the bioreactor, with all sub-types of immature liver-like cells adhered together.

  According to the present disclosure, pluripotent stem cells can be stably differentiated at high cell density and high growth rate.

It is a schematic diagram which shows a general concept. It is a figure which shows a general result. FIG. 5 shows an iPS differentiation protocol. FIG. 1 is a schematic view of a bioreactor design. It is a microscope picture which shows the tissue form in a bioreactor. It is a microscope picture which shows bioreactor staining. FIG. 10 is an image of 3D reconstitution of cells in a bioreactor. Figure 10 is another photomicrograph showing bioreactor staining. It is a graph which shows the result of functional analysis. It is a graph which shows the result of a CYP3A4 and CYP1A2 analysis.

  Embodiments will be described in detail with reference to the drawings.

  FIG. 1 is a schematic view showing a general concept. FIG. 2 is a diagram showing a general result. FIG. 3 shows an iPS differentiation protocol.

  In this embodiment, we propose a new process of differentiation of pluripotent stem cells using biochemical and mechanical stimulation. Pluripotent stem cells that can be differentiated according to the present embodiment are preferably primed cells. Within the category of primed stem cells, pluripotent stem cells such as human iPS cells, human ES cells and human mES cells, induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, mesenchymal system All of the stem cells are included. Other animal sources such as bovine, monkey and rodent stem cells are also suitable. The inventor used only human iPS cells in the present embodiment for the convenience of experiments. In addition, human embryonic stem cells as used herein are obtained by general methods that do not destroy human embryos, such as clonal cultures of human embryonic stem cells (Rodin et al, Nation Communication, June 2014). However, derivation of human stem cell lines from a single blastomere (Klimanskaya et al., Nature, 2006) or other methods can yield human embryonic stem cells without destroying human embryos. Are well known to those skilled in the art. Preferably, induced pluripotent stem cells (iPSCs) are used in the methods of the invention. More preferably, the iPSCs are human iPSCs.

  One of the key points is to stop the general differentiation of iPS in a petri dish used as a container at the immature stage of final tissue-targeted iPS cells. Thus, the iPS population is still in a heterogeneous state comprising several subtypes. The iPS cells are then transferred to the bioreactor and the differentiation protocol is continued. Compared to petri dish culture, the bioreactor environment provides additional stimulation to iPS during the differentiation protocol. Biochemical stimuli contribute to target iPS differentiation to selected tissue targets. At the same time, mechanical stimulation in the bioreactor contributes to direct a portion of the cell population to the second lineage. According to experiments conducted by the inventor, hyperoxia, growth factor gradients and their local concentrations (paracrine or autocrine, endocrine) along the bioreactor improve selective differentiation and hence It was observed that overall tissue maturation was improved (as shown in FIG. 1). Figure 1 is a microscale mimicking in vivo physiology such as (i) shear stress, (ii) oxygen gradient, (iii) iPS subpopulation, (iv) growth factor, and (v) 3D cultured tissue control. To illustrate the general conceptual strategy of experiments based on liver maturation in a bioreactor (biochip). In the case of liver differentiation using this protocol, the inventor has identified intrahepatic, endothelial and epithelial bile such as subdifferentiation lines. The bioreactor and the proposed protocol act as a mimicking microenvironment in vivo (as shown in FIG. 2). Figure 2 illustrates an example of the general results: (A) liver sinusoids, (B) mimic of human iPS liver co-culture, (C) red albumin and green stabilin positive cells, (D) 2.) Albumin production, and (E) CYP450 activity in biochips (bioreactors) and petri (petri dishes or containers).

  Pluripotent stem cells have two major properties, one being that they may differentiate into all types of cells that make up the body (pluripotent) and the other one is that The ability to grow indefinitely in the medium under ad hoc conditions (self-renewal). These cells are thus meant to be a potentially inexhaustible source of mature and functional differentiated cells. At present, it can be considered that human pluripotent cells can be differentiated into hepatocytes, HEP-LC (cells resembling hepatocytes). HEP-LC is a marker of major liver phenotype, showing simulated hepatic metabolism including foreign body metabolism. Despite these promising results, the aforementioned problems still remain. Therefore, the inventor has integrated a further approach to solve these problems. The protocol proposed in this embodiment is based on pre-differentiation in petri dishes and maturation using a bioreactor (as shown in FIG. 3) and a sequence of stimuli. FIG. 3 shows a protocol for differentiation based on seeding of immature iPS into a bioreactor (biochip) after step 3 of petri dish differentiation.

  The method for differentiating human induced pluripotent stem cells according to the present embodiment should not be limited to differentiating into hepatocytes or HEP-LC, but for other organs such as spleen, pancreas, heart and intestine. It should be noted that it could also be used to differentiate into cells of However, for convenience, the object described in the present embodiment is to differentiate human induced pluripotent stem cells into hepatocytes or HEP-LC.

  Next, fabrication of a bioreactor used in the present embodiment, that is, a biochip will be described.

  FIG. 4 is a schematic of the design of the bioreactor.

  Bioreactors (biochips) were fabricated by molding replicates in polydimethylsiloxane (PDMS). The mold was made by a double photolithographic process using SU-8 photoresist. The bioreactor contains a cell culture chamber 5 cm long and 1 cm wide. The height is 300 μm. At the bottom of the culture chamber, an array of microchambers and microchannels is formed to enhance multi-layered cell culture and microfluidic cell culture. As shown in the leftmost view in the middle section of FIG. 1 and in the second view from the left in the lower section of FIG. 3, the cross-sections of the floor surfaces of the microchambers and microchannels are uneven, ie, growth factor Irregularities are alternately connected in the flow direction. Thus, iPS cells can be cultured in three dimensions (3D). In addition, oxygen can be supplied by permeating both the PDMS ceiling and floor panels so that the population of iPS cells rapidly increases. This basic unit of this microarray design is based on the previous work of one of the inventors, Eric Leclerc (see Non-Patent Document 2). Eric Leclerc proposed a bioreactor design (as shown in FIG. 4). The mold was made by LAAS equipment with RTB program. The PDMS bioreactor was manufactured at the laboratory of LIMMS / Sakai. Figure 4 shows the design of the bioreactor: (A) Microstructured layer to make the bioreactor. The top layer is used to spread the medium (culture medium). The bottom layer is used to culture cells in microscale microchambers and microchannels. The maximum depth is 300 μm. (B) Bottom layer details including periodic microstructures replicated along with bioreactor information.

Audrey Legendre, et al. Investigation of the hepatotoxicity of flutamide. Toxicology in Vitro 28 (2014): 1075-1087.

  Next, the preliminary iPS cell differentiation protocol used in the present embodiment will be described.

In the present embodiment, the protocol for iPS differentiation is based on the work of Duncan's group (see Non-Patent Document 3). The iPS cells used for the experiments in the present embodiment were obtained from the University of Tokyo. Duncan's group protocol was modified for the 24 well plates by Professor Miyajima's group (a study conducted by Professor Kido). The inventor used this protocol for the six well plates. Six well petri dishes were coated with Matrigel (Matrigel®) for one hour. After washing with culture medium, iPS cells were seeded in petri dishes. The seeding medium was mTeSR (R) supplemented with anti-apoptotic drug. After 24 hours in seeding medium, growing mTeSR (R) medium was used. The differentiation process started when iPS cells reached 90% of confluence. To that end, iPS cells were exposed for 5 days in RPMI medium supplemented with B27 supplement and 100 ng / mL activin A to form definitive endoderm (step 1: within the upper compartment of Figure 3) See the left arrow on the Then, iPS cells were exposed to 10 ng / mL bFGF and 20 ng / mL BMP4 for 5 days to form a specific liver pattern in RPMI + B27 medium (step 2: second from the left in the upper compartment of FIG. 3) See the arrow). At the end of step 2, cells are exposed to 20 ng / mL HGF in RPMI + B27 medium to reach hepatoblast precursors (step 3: see the third arrow from the left in the top compartment in Figure 3). ). During growth, the medium was replaced daily. In steps 1, 2 and 3, the medium was replaced after 24 hours and 72 hours of culture. The growth steps and step 1 were performed in an incubator with 20% O ₂ and 5% CO ₂ , while steps 2 and 3 were performed under 5% O ₂ .

Karim Si-Tayeb, et al. Highly Efficient Generation of Human Hepatocyte-like Cells from Induced Pluripotent Stem Cells. Hepatology, 2010 January; 51 (1): 297-305.

  Next, iPS culture in a bioreactor used in the present embodiment will be described.

The bioreactor was sterilized by autoclave prior to use. The inner surface was coated with a solution of Matrigel (R) for 1 hour. After washing with culture medium, iPS cells were placed in the bioreactor (step 4: see all figures in the lower section in FIG. 3). At the end of step 3 of differentiation, iPS cells were separated from petri dishes in order to perform liver maturation of iPS in a bioreactor. The seeding density in the bioreactor was doubled in a petri dish to avoid low cell numbers and subsequent dedifferentiation in the bioreactor. Cell adhesion was performed in an incubator with 20% oxygen and in the medium of step 3 (using HGF), to which an anti-apoptotic substrate was added. Once iPS cells were attached, the bioreactor was placed in the perfusion loop. The perfusion loop was a bubble trap where the bioreactor and pump were connected in series. The loop tube was made of PTFE. The loop was filled with 3 mL of step 4 medium. The medium of step 4 was Lonza medium supplemented with a provider's growth factor, and further supplemented with 20 ng / mL of OSM. The flow rate was started at 10-25 μL / min (preferably 20 μL / min) to perform dynamic culture. 1.5 mL of medium was replaced daily during perfusion. The experiments were carried out for 1 week in an incubator with 5% CO ₂ and 20% O ₂ .

  According to the conventional method in which immature hepatocyte-like cells are matured in a petri dish, they are separated into respective groups of subtypes, and the cells of each group are matured separately to the maturation stage, and mature liver is obtained. It was common to assemble a group of cells in the maturation stage to obtain cells resembling cells. On the other hand, according to this embodiment, in the bioreactor, a plurality of subtypes of immature liver-like cells are formed into mature liver-like cells in a form in which all the subtypes of immature liver-like cells adhere together. I was able to mature up to. Liver-like cells include cells resembling hepatocytes, cells resembling endothelium, cells resembling bile and the like. This may be due to the fact that the density of the cells is locally high and the surface area per unit is large, as the cells can be cultured in three dimensions in the microchambers and microchannels of the bioreactor, and It may be attributed to the rapid growth of the population of cells, as it is provided internally in the form of flow smoothly and oxygen is sufficiently supplied through the PDMS panel. The high cell density (millions of cells in several microliters) in the bioreactor chamber contributes to local concentration of growth factors, including autocrine and paracrine factors. Heterogeneous microscale environments (microchannels and microchambers) provide a variety of local microenvironments (such as extension along the endothelial cell wall and reorganization by local shear stress) to which cells can adapt.

  Next, the result of the experiment in the present embodiment will be described. It is shown that the protocol used in the present embodiment contributes to the enhancement of hepatocyte maturation and contributes to the creation of a more functional liver tissue as compared to the conventional petri dish method. First, tissue heterogeneity is explained.

  FIG. 5 is a photomicrograph showing tissue morphology in the bioreactor. FIG. 6 is a photomicrograph showing bioreactor staining. FIG. 7 is an image of 3D reconstitution of cells in the bioreactor. FIG. 8 is another photomicrograph showing bioreactor staining.

  When 96 hours passed from the start of perfusion, the inventor was able to clearly observe the phenotype of the shape resembling cubic hepatocytes in the center of the micro chamber and microchannel of the bioreactor. Elongated cells were observed on the side of the microchannel. After 96 hours of culture, hepatocyte-like cells surrounded by morpho-type fibroblast reticular cells were extensively observed throughout the bioreactor microchannels. After 7 days of perfusion, the tissue created in the bioreactor formed dense 3D like tissue (see FIG. 5). FIG. 5 shows the tissue morphology in the bioreactor, (A) and (B) after adhesion, (C), (D), (E) and (F) after 96 hours of culture (G) shows that after 144 hours of culture.

  Tissue immunostaining showed that hepatocyte-like cells were positive for albumin immunostaining (see FIG. 6). FIG. 6 shows bioreactor staining, where (A), (E) and (I) illustrate cell nuclei, (B) and (F) show stabilin positive cells, (C) and (C) G) shows that of albumin-positive cells, (D) and (H) show combined images of cell nucleus, stabilin and albumin, (J) shows those of phalloidin-positive cells, and (K) is alpha- (L) shows the combined image of phalloidin and alpha-fetoprotein. Stabilin positive cells appeared to surround and embed albumin positive cells in their area. In addition, cells located at the top of the microstructure and tissues were stabilin positive rather than albumin positive (see FIGS. 6 and 7). FIG. 7 shows 3D reconstruction from confocal images of red albumin and green stabilin positive cells in a bioreactor.

  Choryllycylfluorescein (CLF) was secreted into the bile ducts by the bile salt excretion pump (BSEP). Although CLF staining generally indicates a large number of positive cells in the bioreactor medium, a dense biliary ductal network with CLF accumulation was observed in cell clumps that plug the walls of the microstructure (see FIG. 8). ). FIG. 8 shows staining in a bioreactor showing cells that are MDR1, CYP1A1, CLF and BSEP positive. There, DAPI indicates cell nuclei and AFP is weakly expressed. Accumulation similar to the CLF network was slightly observed in the petri dishes. Furthermore, immunostaining of BSEP revealed that bioreactor tissue was positive for this transporter, mainly on the microchannel side. The superposition of BSEP and CLF accumulation confirmed that BSEP and CLF coexisted extensively.

  Using a set of images from several bioreactors and experiments, based on image processing, the inventor established the ratio of albumin positive cells to the total cell population. In the bioreactor up to 60 ± 8% albumin positive cells were found, whereas in the petri dishes only 29 ± 1% albumin positive cells were found. Furthermore, based on the FACS experiments, the inventor observed that in the in-reactor culture, 36 ± 6% of the cells are albumin positive, whereas in the Petri dish culture a greater dispersion is observed (23 ± 23 %) Proved that.

  Next, organizational functionality will be described.

  FIG. 9 is a graph showing the results of functional analysis. FIG. 10 is a graph showing the results of CYP3A4 and CYP1A2 analysis.

  The levels of glucose consumption and lactate formation were assessed to obtain information on respiratory and glycolytic status during culture (see FIG. 9). FIG. 9 shows functional basic analysis in bioreactors and petri dishes. (A) shows lactate glucose ratio, (B) shows albumin production, (C) shows alpha-fetoprotein, (D) shows albumin / alpha-fetoprotein ratio. After cell adhesion and perfusion for 24 hours, in the bioreactor, the inventor discovered intense glucose consumption with high lactate / glucose ratio, indicating anaerobic respiration. After 48 hours of perfusion, the lactate / glucose ratio maintained a value less than 1 close to 0.8, indicating healthy aerobic respiration throughout the tissue. The anaerobic profile was observed to have a ratio of values in the range between 1.6 and 1.8 in Petri dish culture when albumin production is high.

  In order to assess the functionality of the cells with respect to the level of maturation in the bioreactor and petri dish, we measured the production of albumin and alpha-fetoprotein. When the measurements were normalized by the number of hepatocytes in each culture, it was found that bioreactor production was higher in bioreactor production than in petri dishes. At the same time, the kinetics of the AFP / ALB ratio decreased with time for the bioreactor, indicating continuous maturation of hepatocytes in the bioreactor (see FIG. 9).

  To confirm the level of maturation of iPS hepatocyte-like cells in the bioreactor and the functional performance of the tissue, we performed CYP3A4 and CYP1A2 analysis (see FIG. 10). FIG. 10 (A) shows the activity of CYP3A4 and CYP1A2 measured by the formation of luciferin from a dedicated substrate, and FIG. 10 (B) shows typical luciferin production by CYP3A4 following induction by rifampicin in a bioreactor It shows. To that end, the inventor investigated luciferin production from specific substrates in bioreactors and petri dishes. In all Petri dish cultures, we did not detect luciferin production. In contrast, luciferin was generated from iPS cells cultured in a bioreactor. Furthermore, treatment with 25 μm rifampicin contributed to induce the cells. In fact, compared to untreated cells, treated cells doubled the production of luciferin. Petri dish culture was not inducible.

  Next, the effects provided by the present embodiment will be described.

  One of the features of this embodiment is that iPS cells can be differentiated as microscale bioartificial organs in combination with bioreactor and bioreactor technology. Many groups are developing tissue-engineered processes to provide a better environment for the maintenance and development of hepatocytes. This environment should reproduce the characteristics found in vivo as precisely as possible. One such in vitro system can be made using the recent advances in microtechnology to design microscale in vivo simulators. The cell reorganization brought about by the microtopography of these systems, plus dynamic microfluidic culture conditions, appears to be the main feature replicating 3D multicells in vitro. As an example of a possible method, the inventor has shown preliminary results of hepatic iPS differentiation in a bioreactor. Multicellular differentiation and early hepatocyte maturation were achieved in the bioreactor. In addition, a liver-like span in the bioreactor coupled with functional CYP450 activity was observed when compared to petri dishes. Although the point of view of the bioreactor is important, this embodiment involves iPS pre-treatment using conventional petri dish predifferentiation to obtain a heterogeneous and immature subpopulation of iPS cells.

  In this embodiment, chemical stimulation by endocrine growth factor, gradient of growth factor along cell culture area, mechanical stimulation by shear stress, oxygen regulation by permeable material used as support for cells, and surface It includes several novel features such as iPS differentiation protocols that use multiple stimuli including 3D cell reorganization with ultrastructural support that allows for high cell density and large volumes of cell culture area. All these features can be included in the protocol using a microfluidic bioreactor.

  Existing techniques and protocols promote iPS differentiation by adding specific growth factors in the culture medium in well defined sequences or by genetic recombination. The approach of the present embodiment provides an additional type of stimulation to trigger other cellular pathways involved in stem cell differentiation.

  Conventional protocols have not led to hepatic hepatocytes for maturation. Similarly, when applied to other tissues such as the pancreas, the generated cells were weak and functional compared to human mature tissues.

  Next, utilization of the present embodiment will be described.

  The current status of the iPS pattern primitives of the liver is one drawback in putting the treatment solution of iPS of the liver into clinical trials. The limited usefulness of functional hepatocytes for drug testing is also reported as a major obstacle to causing pharmaceutical companies to spend $ 1 billion annually on hepatocytes alone. The ability of the future to create a functional supply of hepatocytes from human pluripotent stem cells can alter this situation. Currently, the scale of the bioreactor used in this embodiment is too small to think about large-scale generation of functional cells for liver transplantation. The main application is currently drug screening assays, whereby it is a partial substitute for human primary cells in drug assays.

  The concept of a protocol using partially differentiated iPS cells prior to culture in a bioreactor that mimics in vivo physiology can be applied to many other target organs such as pancreas, intestine, kidney . Other applications may be more broadly related to regenerative and customized medicine. In fact, based on the patient's collection of iPS cells, the particular cell therapy or disease associated with the patient may be more appropriately investigated. Ultimately, if the protocol could be extended to large scale production, it would be possible to generate sufficient cells for tissue and organ transplantation.

  The disclosure in the present specification is not limited to the above embodiment, and may be variously modified and changed. Therefore, modifications and variations are not excluded from the scope of protection of the claims appended hereto.

  The present invention is applicable to methods of differentiating pluripotent stem cells.

Claims (8)

A method of differentiating pluripotent stem cells:
Seeding the pluripotent stem cells in a container comprising a seeding medium;
Differentiating the pluripotent stem cells in the container;
Transferring the pluripotent stem cells from the container into the chamber of the bioreactor when the pluripotent stem cells reach the precursor stage;
Maturing said pluripotent stem cells in said chamber;
The method wherein the floor of the chamber includes a recess or a protrusion and the fluid of the culture medium flows in the chamber and oxygen is supplied to the chamber.   The method according to claim 1, wherein the bioreactor is mounted in a perfusion loop and connected to a pump in the perfusion loop, the perfusion loop being filled with medium.   The method of claim 2, wherein the bottom of the chamber comprises an array of microchambers and microchannels.   The method according to claim 1, wherein the pluripotent stem cells are human induced pluripotent stem cells.   The method according to claim 4, wherein the human induced pluripotent stem cells are differentiated into HEP-LC.   6. The method according to claim 5, wherein the precursor stage is a stage in which definitive endoderm is formed, a stage in which a specific liver pattern is formed, or a stage in which immature hepatoblasts are formed.   5. The method according to claim 4, wherein the human induced pluripotent stem cells are transferred from the container into the chamber of the bioreactor in a form in which all sub-types of immature liver-like cells adhere together.   8. The method according to claim 7, wherein the human induced pluripotent stem cells are matured in the chamber of the bioreactor in a form in which all sub-types of immature liver-like cells are adhered together.

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